Biology subjects

Campos, J.

Publications and source records attributed to Campos, J..

30 records · Page 2Linked to original sources

bioRxiv2023

The phenuivirus Toscana virus makes an atypical use of vacuolar acidity to enter host cells

Toscana virus is a major cause of arboviral disease in humans in the Mediterranean basin during summer. However, early virus-host cell interactions and entry mechanisms remain poorly characterized. Investigating iPSC-derived human neurons and cell lines, we found that virus binding to the cell surface was specific but inefficient, and 50% of bound virions were endocytosed within 10 min. Virions entered Rab5a+ early endosomes and, subsequently, Rab7a+ and LAMP-1+ late endosomal compartments. Penetration required intact late endosomes and occurred within 30 min following internalization. Virus entry relied on vacuolar acidification, with an optimal pH for viral membrane fusion at pH 5.5. The pH threshold increased to 5.8 with longer pre-exposure of virions to the slightly acidic pH in early endosomes. Strikingly, the particles remained infectious after entering late endosomes with a pH below the fusion threshold. Overall, our study establishes Toscana virus as a late-penetrating virus and reveals an atypical use of vacuolar acidity by this virus to enter host cells.

microbiology↗

bioRxiv2022

Transcriptomic cytoarchitecture reveals principles of human neocortex organization

Variation in cortical cytoarchitecture is the basis for histology-based definition of cortical areas, such as Brodmann areas. Single cell transcriptomics enables higher-resolution characterization of cell types in human cortex, which we used to revisit the idea of the canonical cortical microcircuit and to understand functional areal specialization. Deeply sampled single nucleus RNA-sequencing of eight cortical areas spanning cortical structural variation showed highly consistent cellular makeup for 24 coarse cell subclasses. However, proportions of excitatory neuron subclasses varied strikingly, reflecting differences in intra- and extracortical connectivity across primary sensorimotor and association cortices. Astrocytes and oligodendrocytes also showed differences in laminar organization across areas. Primary visual cortex showed dramatically different organization, including major differences in the ratios of excitatory to inhibitory neurons, expansion of layer 4 excitatory neuron types and specialized inhibitory neurons. Finally, gene expression variation in conserved neuron subclasses predicts differences in synaptic function across areas. Together these results provide a refined cellular and molecular characterization of human cortical cytoarchitecture that reflects functional connectivity and predicts areal specialization.

neuroscience↗

bioRxiv2022

A time and single-cell resolved model of hematopoiesis

The paradigmatic tree model of hematopoiesis is increasingly recognized to be limited as it is based on heterogeneous populations and largely inferred from non-homeostatic cell fate assays. Here, we combine persistent labeling with time-series single-cell RNA-Seq to build the first real- time, quantitative model of in vivo tissue dynamics for any mammalian organ. We couple cascading single-cell expression patterns with dynamic changes in differentiation and growth speeds. The resulting explicit linkage between single cell molecular states and cellular behavior reveals widely varying self-renewal and differentiation properties across distinct lineages. Transplanted stem cells show strong acceleration of neutrophil differentiation, illustrating how the new model can quantify the impact of perturbations. Our reconstruction of dynamic behavior from snapshot measurements is akin to how a Kinetoscope allows sequential images to merge into a movie. We posit that this approach is broadly applicable to empower single cell genomics to reveal important tissue scale dynamics information. HighlightsO_LICell flux analysis reveals high-resolution kinetics of native bone marrow hematopoiesis C_LIO_LIQuantitative model simulates cell behavior in real-time and connects it with gene expression patterns C_LIO_LIDistinct lineage-affiliated progenitors have unique self-renewal and differentiation properties C_LIO_LITransplanted HSCs display accelerated stage- and lineage-specific differentiation C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/506735v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@244ad6org.highwire.dtl.DTLVardef@ad632borg.highwire.dtl.DTLVardef@149daf9org.highwire.dtl.DTLVardef@1c7183f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Condensin I folds the C. elegans genome

The Structural Maintenance of Chromosomes (SMC) complexes, cohesin and condensins, are named for their roles in separating and compacting chromosomes during meiosis and mitosis. Recent data from mammalian cells have revealed additional functions for cohesin, including folding the interphase genome into loops and domains. However, it remains unclear what determines genome folding in holocentric species. To address this question, we systematically and acutely inactivated each SMC complex. Surprisingly, we found that, in contrast to mammals, condensin I is the major long-range genome loop extruder, while cohesin only creates small loops. Specifically, loss of condensin I led to genome-wide decompaction, chromosome mixing, and the disappearance of topologically associating domain (TAD) structures, while reinforcing fine-scale epigenomic compartments. Strikingly, inactivating condensin I and its X-specific variant condensin IDC from the X chromosomes revealed the existence of a third compartment that groups together a subset of previously characterized loading sites for condensin IDC and binding sites for the X-targeting complex SDC. Although the inactivation of cohesin, condensin II, and condensin I/IDC led to minor transcriptional changes for all autosomes, removing condensin I/IDC from the X chromosome resulted in the up-regulation of X-linked genes. In conclusion, our findings describe a novel function for C. elegans condensin I/IDC in organizing holocentric interphase chromosomes, which substitutes for the role played by cohesin in mammals.

molecular biology↗

bioRxiv2022

SARS-CoV-2 Omicron potently neutralized by a novel antibody with unique Spike binding properties

The SARS-CoV-2 Omicron variant exhibits very high levels of transmission, pronounced resistance to authorized therapeutic human monoclonal antibodies and reduced sensitivity to vaccine-induced immunity. Here we describe P2G3, a human monoclonal antibody (mAb) isolated from a previously infected and vaccinated donor, which displays picomolar-range neutralizing activity against Omicron BA.1, BA.1.1, BA.2 and all other current variants, and is thus markedly more potent than all authorized or clinically advanced anti-SARS-CoV-2 mAbs. Structural characterization of P2G3 Fab in complex with the Omicron Spike demonstrates unique binding properties to both down and up spike trimer conformations at an epitope that partially overlaps with the receptor-binding domain (RBD), yet is distinct from those bound by all other characterized mAbs. This distinct epitope and angle of attack allows P2G3 to overcome all the Omicron mutations abolishing or impairing neutralization by other anti-SARS-COV-2 mAbs, and P2G3 accordingly confers complete prophylactic protection in the SARS-CoV-2 Omicron monkey challenge model. Finally, although we could isolate in vitro SARS-CoV2 mutants escaping neutralization by P2G3 or by P5C3, a previously described broadly active Class 1 mAb, we found these viruses to be lowly infectious and their key mutations extremely rare in the wild, and we could demonstrate that P2G3/P5C3 efficiently cross-neutralized one anothers escapees. We conclude that this combination of mAbs has great prospects in both the prophylactic and therapeutic settings to protect from Omicron and other VOCs.

immunology↗

bioRxiv2021

Fibroblastic reticular cells provide a supportive niche for lymph node-resident macrophages

The lymph node (LN) is home to resident macrophage populations that are essential for immune function and homeostasis. The T cell paracortical zone is a major site of macrophage efferocytosis of apoptotic cells, but key factors controlling this niche are undefined. Here we show that fibroblastic reticular cells (FRCs) are an essential component of the LN macrophage niche. Macrophages co-localised with FRCs in human LNs, and murine single-cell RNA-sequencing revealed that most reticular cells expressed master macrophage regulator CSF1. Functional assays showed that CSF1R signalling was sufficient to support macrophage development. In the presence of LPS, FRCs underwent a mechanistic switch and maintained support through CSF1R-independent mechanisms. These effects were conserved between mouse and human systems. Rapid loss of macrophages and monocytes from LNs was observed upon genetic ablation of FRCs. These data reveal a critically important role for FRCs in the creation of the parenchymal macrophage niche within LNs.

immunology↗

bioRxiv2021

Mild impairment of mitochondrial function increases longevity and pathogen resistance through ATFS-1-driven activation of p38-regulated innate immunity

While mitochondrial function is essential for life in all multicellular organisms, a mild impairment of mitochondrial function can extend longevity. By understanding the molecular mechanisms involved, these pathways might be targeted to promote healthy aging. In studying two long-lived mitochondrial mutants in C. elegans, we found that disrupting subunits of the mitochondrial electron transport chain resulted in upregulation of genes involved in innate immunity, which we found to be dependent on not only the canonical p38-mediated innate immune signaling pathway but also on the mitochondrial unfolded protein response. Both of these pathways are absolutely required for the increased resistance to bacterial pathogens and extended longevity of the long-lived mitochondrial mutants, as is the FOXO transcription factor DAF-16. This work demonstrates that both the p38-mediated innate immune signaling pathway and the mitochondrial unfolded protein response can act on the same innate immunity genes to promote resistance to bacterial pathogens, and that input from the mitochondria can extend longevity by signaling through these two pathways. Combined, this indicates that multiple evolutionarily conserved genetic pathways controlling innate immunity also function to modulate lifespan. Significance StatementIn this work, we explore the relationship between mitochondrial function, aging and innate immunity. We find that mild impairment of mitochondrial function results in upregulation of genes involved in innate immunity, increased resistance to bacterial pathogens and lifespan extension, all of which are dependent on two evolutionarily conserved signaling pathways. This work demonstrates how changes in functional status of the mitochondria can trigger activation of innate immunity, and that the underlying mechanisms are important for the longevity of the organism. This work advances our understanding of connections between metabolism and immunity. As the pathways studied here are conserved up to mammals, these insights may help us to understand the role of mitochondrial health, innate immunity and lifespan in humans.

cell biology↗

bioRxiv2020

CLEC-2 promotes inflammatory peritoneal macrophage emigration to draining lymph nodes during endotoxemia

Macrophage recruitment during sterile inflammation and infection is essential to clear pathogens, apoptotic cells and debris. However, persistent macrophage accumulation leads to chronic inflammation. Platelets are emerging as key modulators of the inflammatory response. Here, we identify that platelet C-type-lectin-like receptor-2 (CLEC-2) is a crucial immunomodulatory receptor through the interaction with podoplanin, upregulated on inflammatory macrophages. Mechanistically, platelet CLEC-2 upregulates the expression of podoplanin and its co-ligands CD44 and ERM proteins, leading to actin rearrangement and promotion of cell migration; this is mimicked by recombinant CLEC-2-Fc (rCLEC-2-Fc). Treatment of LPS-challenged mice with rCLEC-2-Fc induces a rapid emigration of peritoneal macrophages to mesenteric lymph nodes, through a gradient generated by the podoplanin ligand, CCL21, to prime T cells. We propose that crosslinking podoplanin using rCLEC-2-Fc is a novel, cell-specific strategy to accelerate macrophage removal from the site of inflammation, and hence promote the resolution of the inflammatory response. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/423770v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@e37acborg.highwire.dtl.DTLVardef@92a5e1org.highwire.dtl.DTLVardef@1c8832eorg.highwire.dtl.DTLVardef@11c5c97_HPS_FORMAT_FIGEXP M_FIG C_FIG SummaryPersistent macrophage accumulation in inflamed tissue leads to chronic inflammation and organ damage. Bourne et al. identify recombinant CLEC-2-Fc crosslinking podoplanin on inflammatory macrophages, as a cell-specific strategy to accelerate their emigration to draining lymph nodes, and reduce local inflammation.

immunology↗

bioRxiv2020

BRD4-mediated repression of p53 is a target for combination therapy in AML

Acute Myeloid Leukemia (AML) is a typically-lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, most AML retain wild-type TP53, encoding the pro-apoptotic tumor suppressor p53. MDM2 inhibitors (MDM2i), which activate wild-type p53, and BET inhibitors (BETi), targeting the BET-family co-activator BRD4, both show encouraging pre-clinical activity, but limited clinical activity as single agents. Here, we report synergistic toxicity of combined MDM2i and BETi towards AML cell lines, primary human blasts and mouse models, resulting from BETis ability to evict an unexpected repressive form of BRD4 from p53 target genes, and hence potentiate MDM2i-induced p53 activation. These results indicate that wild-type TP53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML.

cell biology↗

bioRxiv2020

Bi-allelic variants in TSPOAP1, encoding the active zone protein RIMBP1, cause autosomal recessive dystonia

Dystonia is a debilitating hyperkinetic movement disorder, frequently transmitted as a monogenic trait. Here, we describe homozygous frameshift, nonsense and missense variants in TSPOAP1, encoding the active zone RIM-binding protein 1 (RIMBP1), as a novel genetic cause of autosomal recessive dystonia in seven subjects from three unrelated families. Subjects carrying loss-of-function variants presented with juvenile- onset progressive generalized dystonia, associated with intellectual disability and cerebellar atrophy. Conversely, subjects carrying a pathogenic missense variant (p.Gly1808Ser) presented with isolated adult-onset focal dystonia. In mice, complete loss of RIMBP1, known to reduce neurotransmission, led to motor abnormalities reminiscent of dystonia, decreased Purkinje cell dendritic arborization, and reduced numbers of cerebellar synapses. In vitro analysis of the p.Gly1808Ser variant showed larger spike-evoked calcium transients and enhanced neurotransmission, suggesting that RIMBP1-linked dystonia can be caused by either reduced or enhanced rates of spike-evoked release in relevant neural networks. Our findings establish a direct link between presynaptic RIMBP1 dysfunction and dystonia and highlight the critical role played by well-balanced neurotransmission in motor control and disease pathogenesis.

genetics↗

bioRxiv2019

VE-cadherin endocytosis controls vascular integrity and patterning during development

Tissue morphogenesis requires dynamic intercellular contacts that are subsequently stabilized as tissues mature. The mechanisms governing these competing adhesive properties are not fully understood. Using gain- and loss-of-function approaches, we tested the role of p120-catenin (p120) and VE-cadherin (VE-cad) endocytosis in vascular development using mouse mutants that exhibit increased (VE-cadGGG/GGG) or decreased (VE-cadDEE/DEE) internalization. VE-cadGGG/GGG mutant mice exhibited reduced VE-cad-p120 binding, reduced VE-cad levels, microvascular hemorrhaging, and decreased survival. By contrast, VE-cadDEE/DEE mutants exhibited normal vascular permeability but displayed microvascular patterning defects. Interestingly, VE-cadDEE/DEE mutant mice did not require endothelial p120, demonstrating that p120 is dispensable in the context of a stabilized cadherin. In vitro, VE-cadDEE mutant cells displayed defects in polarization and cell migration that were rescued by uncoupling VE-cadDEE from actin. These results indicate that cadherin endocytosis coordinates cell polarity and migration cues through actin remodeling. Collectively, our results indicate that regulated cadherin endocytosis is essential for both dynamic cell movements and establishment of stable tissue architecture.\n\nSummary StatementThis study uses mouse genetic and in vitro approaches to demonstrate that cadherin endocytosis is critical for the formation of blood vessels during development by promoting actin-dependent collective cell migration, whereas the inhibition of this endocytosis by p120 binding is essential for vessel stabilization.

cell biology↗

bioRxiv2018

Pathologically distinct fibroblast subsets drive inflammation and tissue damage in arthritis

The identification of lymphocyte subsets with non-overlapping effector functions has been pivotal to the development of targeted therapies in immune mediated inflammatory diseases (IMIDs). However it remains unclear whether fibroblast subclasses with non-overlapping functions also exist and are responsible for the wide variety of tissue driven processes observed in IMIDs such as inflammation and damage. Here we identify and describe the biology of distinct subsets of fibroblasts responsible for mediating either inflammation or tissue damage in arthritis. We show that deletion of FAP+ synovial cells suppressed both inflammation and bone erosions in murine models of resolving and persistent arthritis. Single cell transcriptional analysis identified two distinct fibroblast subsets: FAP+ THY1+ immune effector fibroblasts located in the synovial sub-lining, and FAP+ THY1- destructive fibroblasts restricted to the synovial lining. When adoptively transferred into the joint, FAP + THY1- fibroblasts selectively mediate bone and cartilage damage with little effect on inflammation whereas transfer of FAP + THY1+ fibroblasts resulted in a more severe and persistent inflammatory arthritis, with minimal effect on bone and cartilage. Our findings describing anatomically discrete, functionally distinct fibroblast subsets with non-overlapping functions have important implications for cell based therapies aimed at modulating inflammation and tissue damage.

immunology↗